The ability to detect DNA sequence variances (hereinafter referred to as “polymorphisms” for convenience, but without limitation) in an organism's genome has become an important tool in the diagnosis of diseases and disorders and in the prediction of response to therapeutic regimes. It is becoming increasingly possible, using early variance detection, to diagnose and treat, even prevent the occurrence of, a disease or disorder before it has physically manifested itself. Furthermore, variance detection can be a valuable research tool in that it may lead to the discovery of genetic bases for disorders the cause of which were hitherto unknown or thought to be other than genetic.
Sequence variance can take a number of different forms. For example, a variance can arise through substitution of one or more nucleotides for the same number of others at a particular locus in a gene. Another example would be the deletion of one or more nucleotides from a particular locus in a gene. A still further example would be the insertion of one or more extra nucleotides at a particular locus in a gene. Combinations of substitution, deletion and insertion are also possible. A common type of sequence variance is the single nucleotide polymorphism or SNP. A SNP involves the substitution of one nucleotide for another at a particular locus in a gene. Although each SNP involves only one nucleotide, a single gene may contain numerous SNPs.
Determination of whether a particular gene of a species or of an individual of that species contains a sequence variance is called genotyping. Complete sequencing is, therefore, a method for accomplishing genotyping but it is slow, costly and extremely inefficient.
An alternative genotyping approach known in the art uses makes use of so-called induced heteroduplex generators (previously also termed universal heteroduplex generators and referred to hereinafter by the term “IHG”). These are synthetic DNA sequences that mimic a genomic DNA sequence, but which contain controlled nucleotide substitutions, deletions, insertions or combinations thereof (collectively termed “identifiers”) engineered at nucleotide positions opposite to and—in the art—contiguous with (that is immediately adjacent to) known polymorphic sites within the genomic DNA. To detect sequence variances within genomic DNA, the IHG and genomic DNA sequences are amplified separately with the same locus-specific PCR primers and the respective PCR products are subsequently hybridised together by heating and slow cooling in order to generate DNA heteroduplexes. The resulting heteroduplexes are then resolved, e.g. by non-denaturing polyacrylamide minigel electrophoresis. Depending on the number, type and position of the mismatches between the IHG and genomic DNA, different heteroduplexes are generated which migrate at different rates, thus giving rise to characteristic banding patterns for different alleles.
The use of induced heteroduplex generators is discussed in the following references:    1. Bidwell, J L, Clay, T M, Wood, N A P, Pursall, M C, Martin, A F, Bradley, B A and Hui, K M (1993) Rapid HLA-DR-Dw and DP matching by PCR fingerprinting and related DNA heteroduplex technologies. pp 99-116 in: Handbook of HLA Typing Techniques [Eds K M Hui & J L Bidwell] CRC Press: Boca Raton, Fla.    2. Wood, N, Tyfield, L, Bidwell, J (1993) Rapid classification of phenylketonuria genotypes by analysis of heteroduplexes generated by PCR-amplifiable synthetic DNA. Human Mutation 2: 131-137.    3. Bidwell, J L, Wood, N A P, Tyfield, L A, Clay, T M, Culpan, D, Evans, J M, Pursall, M C, Bradley, B A (1993) Universal heteroduplex generators: reagents for genotyping of HLA and of human diseases. Molecular Bases of Human Diseases (Ed. E. E. Polli). pp 27-34. Elsevier/North-Holland, Amsterdam.    4. Wood, N, Standen, G, Hows, J, Bradley, B, Bidwell, J. (1993) Diagnosis of sickle cell disease with a universal heteroduplex generator. Lancet 342: 1519-1520.    5. Bidwell, J L, Wood, N A P, Clay, T M, Pursall, M C, Culpan, D, Evans, J, Bradley, B A, Tyfield, L A, Standen, G, Hui, K M (1994) DNA heteroduplex technology. In: Chrambach, A, Dunn, M J, Radola, B J (Eds), Advances in Electrophoresis, Volume 7, pp 311-351. VCH Press, Weinheim.    6. Clay, T M, Cuplan, D, Howell, W M, Sage, D A, Bradley, B A, Bidwell, J L. (1994) UHG crossmatching: a comparison with PCR-SSO typing in the selection of HLA-DPB1-compatible bone marrow donors. Transplantation 58: 200-207.    7. Bidwell, J L. (1994) Advances in DNA-based HLA-typing methods. Immunology Today 15: 303-307.    8. Tyfield, L A, Stephenson, A, Bidwell, J L, Wood, N A P, Cockburn, F, Harvie, A, Smith, I. (1994) Mutation analysis of the phenylalanine hydroxylase gene using heteroduplex analysis with synthetic DNA constructs. Acta Paediatrica 83: 47-48.    9. Savage, D A, Wood, N A P, Bidwell, J L, Hui, K M. (1995) HLA-DRB1*01 subtyping by heteroduplex analysis. Tissue Antigens 45: 120-124.    10. Wood, N, Standen, G, Murray, E W, Lillicrap, D, Holmberg, L, Peake, I R, Bidwell, J. (1995) Rapid genotype analysis in type 2B von Willebrand's disease using a universal heteroduplex generator. Brit J Haematol 89: 152-156.    11. Wood, N, Standen, G, Old, J, Bidwell, J. (1995) Optimisation and properties of a UHG for genotyping of haemoglobins S and C. Hum Mutation 5: 166-172.    12. Savage, D A, Wood, N A P, Bidwell, J L, Fitches, A, Old, J M, Hui, K M. (1995) Detection of b-thalassaemia mutations using DNA heteroduplex generator molecules. Brit J Haematol 90: 564-571.    13. Tyfield, L A, Zschocke, J, Stephenson, A, Cockburn, F, Harvie, A, Bidwell, J L, Wood, N A P, Hunt, L P. (1995) Discordant phenylketonuria phenotypes in one family: the relationship between genotype and clinical outcome is a function of multiple effects. J Med Genet 32: 132-136.    14. Wood, N A P, Bidwell, J L. (1996) UHG: heteroduplex and universal heteroduplex generator analysis. In: Laboratory protocols for mutation detection (ed Landegren, U), pp 105-112. Oxford University Press for the Human Genome Organisation.    15. Wood, N, Bidwell, J. (1996) Genetic screening and testing by induced heteroduplex formation. Electrophoresis 17: 247-254.    16. Wood, N, Standen, G R, Bowen, D J, Cumming, A, Lush, C, Lee, R, Bidwell, J. (1996) UHG-based mutation screening in type 2B von Willebrand's disease: detection of a candidate mutation Ser547Phe. Thrombosis & Haemostasis 75: 363-367.    17. Savage, D A, Tang, J P, Wood, N A P, Evans, J, Bidwell, J L, Wee, J L K, Oei, M, Hui, K M (1996) A rapid HLA-DRB1*04 subtyping method using PCR and DNA heteroduplex generators. Tissue Antigens 47: 284-292.    18. Jack, D, Bidwell, J, Turner, M, Wood, N. (1997) Simultaneous genotyping for all three known structural mutations in the human mannan-binding lectin gene. Human Mutation 9: 41-46.    19. Culpan, D, Standen, G R, Wood, N, Mazurier, C, Gaucher, C, Bidwell, J L (1997) Rapid mutation screening in Type 2A von Willebrand's disease using universal heteroduplex generators. Brit J Haematol 96: 464-469.    20. Bowen, D, et al (1997) Genetic diagnosis of Factor V Leiden using heteroduplex technology. Thrombosis and Haemostasis 77: 119-122.    21. Jackson, H A, Bowen, D J, Worwood, M (1997) Rapid genetic screening for haemochromatosis using heteroduplex technology. Br J Haematol 98: 856-859.    22. Tyfield, L A, Stephenson, A, Cockburn, F, Harvie, A, Bidwell, J L, Wood, N A P, Pilz, D T, Harper, P, Smith, I (1997) Sequence variation at the phenylalanine hydroxylase gene in the British Isles. Am J Hum Genet 60: 388-396    23. Enayat, M S, Theophilus, B D M, Hill, F G H, Rose, P E, Culpan, D, Bidwell, J, Standen, G R (1998) A new candidate mutation (K755E) causing Type 2A Von Willebrand's disease identified by UHG analysis. Br J Haematol 79:240    24. Culpan, D, Goodeve, A, Bowen, D J, Standen, G, Bidwell, J (1988) Rapid genotypic diagnosis of type 2A von Willebrand's disease by heteroduplex analysis. Clin. Lab. Haem 20: 177-178.    25. Bowen, D J, Standen, G R, Mazurier, C, Gaucher, C, Cumming, A, Keeney, S, Bidwell, J (1998) Type 2N von Willebrand disease: rapid genetic diagnosis of G2811A (R854Q), C2696T (R816W), T2701A (H817Q) and G2823T (C858F)-detection of a novel candidate type 2N mutation: C2801T (R854W). Thromb Haemost 80: 32-36.    26. Morse, HR, Olomolaiye, O O, Wood, N A P, Keen, L J and Bidwell, J L (1999) Induced heteroduplex genotyping of TNFa, IL-1b, IL-6 and IL-10 polymorphisms associated with transcriptional regulation. Cytokine 11: 789-795.    27. Wood, N A P, Thompson, S C, Smith, R M, Bidwell, J L (2000) Identification of human TGF-b1 signal (leader) sequence polymorphisms by PCR-RFLP. J Immunol Methods 234: 117-122.    28. Bidwell, J L, Olomolaiye, O O, Keen, L J, Wood, N A P, Morse, H R, Laundy, G J, Thompson, S J (2000) Cytokine gene polymorphism. In: Human Blood Cells: Consequences of Genetic Polymorphism (Ed. King, M-J), pp 375-400. Imperial College Press, London.    29. Wood, N A P, Keen, L J, Tilley, L A, Bidwell, J L (2001) Determination of cytokine regulatory haplotypes by induced heteroduplex analysis of DNA. J Immunol Meth 249:191-198.    30. Spink, C F, Keen, L J, Middleton, P G, Bidwell, J L (2004) Discrimination of suballeles present at the TNFd microsatellite locus using Induced Heteroduplex Analysis. Genes and Immunity 5: 76-79.    31. Smith A J P, Keen L J, Wood N A P, Elson C J, Bidwell J L (2004) Haplotype analysis of IL1A, IL1B, IL1RN and IL1R1 promoter SNPs. In: HLA 2002. Blackwell Munksgaard, Denmark    32. PhD thesis of NAP Wood dated November 1995 entitled “An Investigation of the Potential of Universal Heteroduplex Generators in Identification of Point Mutations Within DNA”, held in the library of the University of Bristol.
WO-A-93/19201 and GB-A-2338062 also relate to genotyping using IHG technology.
The content of the above patent documents and other references is incorporated herein by reference for all purposes.
However, it is to be noted that the content of the above patent documents and other references is not necessarily prior art in every designated state of this PCT patent application, as the prior art status is to be judged according to domestic legislation. Therefore, no admission is made that any particular document referred to herein is prior art to any of the claims presently included in this application, or which may be introduced by future amendment, or which may be included or introduced in any continuation or divisional application, or any other corresponding or further application, which may be filed in the future based on the present PCT patent application.
Whilst genotyping using IHGs is simple to use, may be carried out quickly, and is reliable, it would nevertheless be desirable to improve heteroduplex band resolution, so that the allele-specific patterns are more easily distinguished. This will enable, for example, a shortened gel electrophoresis time.